Streptococcus thermophilus is widely used for the manufacture of yoghurt and Swiss or Italian-type cheeses. These products have a market value of approximately $40 billion per year, making S. thermophilus a species that has major economic importance. Even though the fermentation properties of this bacterium have been gradually improved by selection and classical methods, there is great potential for further improvement through natural processes or by genetic engineering. To be able to improve naturally, without any genetic engineering technology involved, S. thermophilus technological performances, physiological properties, behaviour in different growth media is of key importance for fermented food and feed industries. The obtained improved strains of S. thermophilus will be in total compliance with all current regulations concerning living microorganisms for food and feed.
In silico analyses of the currently available S. thermophilus genome sequences suggest that many genes were acquired through horizontal gene transfer (1). These horizontal gene transfers potentially occurred through natural transformation designated as competence, as reported in pathogenic streptococci that use competence as a general mechanism for genomic plasticity.
Competence is the ability of a cell to take up extracellular (“naked”) DNA from its environment and to integrate this DNA (or part thereof) in its genome. Competence can be separated into natural competence and artificial competence. Natural competence is a genetically specified ability of bacterial strain to perform this DNA uptake. It is thought to occur spontaneously in natural environment and is observed as well in laboratory conditions. Natural competence seems to be a common trait among streptococci (2, 3). By contrast, artificial competence is provoked in laboratory conditions and consists of rendering the cells transiently permeable to naked DNA.
Natural genetic transformation is the process by which a given bacterial strain can be transformed, i.e. modified to integrate a polynucleotide in its genome, through natural competence.
Streptococcal natural competence has been most extensively studied in Streptococcus pneumoniae, and appears to be a common feature to streptococci species. Competence is generally tightly regulated and involves two sets of genes: the early and the late competence genes (4). In S. pneumoniae, natural competence is a transient event that relies on the accumulation of a pheromone (CSP) in the growth medium. The early competence genes encode a two-component system (TCS: ComD and ComE), its cognate induction factor (CSP: ComC), and the components of an ABC transporter dedicated to CSP export and maturation (ComA and ComB). Upon CSP induction, TCS activates transcription of the competence sigma factor ComX (σX). This alternative sigma factor is the central regulator of natural competence since it positively regulates all essential late genes necessary for DNA uptake, protection and integration into the chromosome (4). See FIG. 1 for schematic representation.
A putative comX gene and other genes resembling all late competence genes essential for S. pneumoniae natural competence were found in the S. thermophilus genome (1). Moreover, putative “ComX-boxes” recognized by σX were in silico-identified upstream of all operons containing essential late competence genes. This maintenance of all essential genes for natural competence in S. thermophilus suggested that they play a key physiological function (use of DNA as a nutrient, genome plasticity . . . ) in its ecological niche (1). In 2006, the group of Havarstein (5) has shown the functionality of natural competence in S. thermophilus LMG18311 through the use of genetic engineering methodologies. This was achieved by using a plasmid-based system containing comX under the control of a regulated peptide-induced promoter (blp system, (6)). Remarkably, high transformation frequency (10−3 to 10−2) was achieved with an efficacy similar to S. pneumoniae with either total genomic DNA or linear dsDNA (5). It is noticeable that in this case, induction of late competence genes was artificially made possible through the genetically engineered induction of ComX. However and surprisingly, none of the early competence genes usually found in S. pneumoniae genomes and that play a crucial role in the induction of natural competence are present in S. thermophilus genomes. Recently, it was demonstrated that competence in S. thermophilus LMD-9 can be induced during growth in a chemically defined medium. In strain LMD-9, the transporter Ami was shown to be specifically required for the transcriptional induction of comX in those conditions (7). The Ami system actively imports oligopeptides present in the extracellular medium and is known to have both nutritive and signaling functions in Gram-positive bacteria (7, 8, 9). Since this chemically defined medium is a peptide-free medium, Gardan et al. (7) hypothesized that, in those growth conditions, the bacterium synthesizes and secretes a specific competence-stimulating peptide, which is then sensed and re-imported by the Ami system. Once internalized, this pheromone would then interact with a specific cytoplasmic regulator, leading to the transcriptional induction of comX (for a model, see FIG. 2). The pheromone and the transcriptional regulator responsible for comX expression and natural transformation in the chemically defined medium conditions are still unknown.
Although natural transformation in chemically defined medium was very efficient in strain LMD-9 (10−4 to 10−3), the transformation rate of strain LMG18311 was remarkably lower (10−7 to 10−6). In addition, no transformant could be detected in the case of strain CNRZ1066 grown in chemically defined medium (transformation rate <10−8) (7). To the inventors' knowledge, there is no successful report of a method to induce natural competence in strains of S. thermophilus without genetic engineering.
There is still a need in the art for a method for inducing natural competence in a Streptococcus bacterium, preferably in a S. thermophilus and/or a S. salivarius bacterium, and for transforming said bacterium, using a technology that is recognized world-wide as non-GMO.